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Kaposi sarcoma (KS) punch biopsies present unique challenges for extracting nucleic acids, which can be exacerbated by their long-term stabilization in RNAlater. Here, we present a protocol for simultaneously isolating DNA, RNA, and miRNA from a single KS punch biopsy. We detail the steps for preparing reagents and supplies, disrupting KS tissue using manual and mechanical methods, isolating DNA and total RNA, evaluating nucleic acid quality, and storing nucleic acids long-term.

BackgroundSex differences in immune-based disorders are well-established, with female sex associated with a markedly heightened risk of autoimmune disease. Female sex is also overrepresented in other conditions associated with elevated inflammation, including depression, chronic pain, and chronic fatigue. The mechanisms underlying these disparities are unclear. This study used an experimental model of inflammatory challenge to interrogate molecular mechanisms that may contribute to female vulnerability to disorders with an inflammatory basis.MethodIn this analysis of a secondary outcome from a randomized controlled trial, 111 participants (67 female) received either a bolus injection of endotoxin (n = 59) or placebo (n = 52). Participants provided blood samples before and 0.5 h post-injection for assessment of differential activation of key pro-inflammatory (i.e., activator protein (AP)-1; nuclear factor (NF)-κB) and immunoregulatory (i.e., glucocorticoid receptor (GR); cAMP response element binding protein (CREB)) signaling pathways via genome-wide expression profiling and promoter-based bioinformatics analyses.ResultsRelative to males, females exhibited greater endotoxin-induced increases in bioinformatic measures of CREB transcription factor activity (p's

The presence of HIV in sequestered reservoirs is a central impediment to a functional cure, allowing HIV to persist despite life-long antiretroviral therapy (ART), and driving a variety of comorbid conditions. Our understanding of the latent HIV reservoir in the central nervous system is incomplete, because of difficulties in accessing human central nervous system tissues. Microglia contribute to HIV reservoirs, but the molecular phenotype of HIV-infected microglia is poorly understood. We leveraged the unique "Last Gift" rapid autopsy program, in which people with HIV are closely followed until days or even hours before death. Microglial populations were heterogeneous regarding their gene expression profiles but showed similar chromatin accessibility landscapes. Despite ART, we detected occasional microglia containing cell-associated HIV RNA and HIV DNA integrated into open regions of the host's genome (∼0.005%). Microglia with detectable HIV RNA showed an inflammatory phenotype. These results demonstrate a distinct myeloid cell reservoir in the brains of people with HIV despite suppressive ART. Strategies for curing HIV and neurocognitive impairment will need to consider the myeloid compartment to be successful.

BACKGROUND: Phenotypic heterogeneity in early language, intellectual, motor, and adaptive functioning (LIMA) features are amongst the most striking features that distinguish different types of autistic individuals. Yet the current diagnostic criteria uses a single label of autism and implicitly emphasizes what individuals have in common as core social-communicative and restricted repetitive behavior difficulties. Subtype labels based on the non-core LIMA features may help to more meaningfully distinguish types of autisms with differing developmental paths and differential underlying biology. METHODS: Unsupervised data-driven subtypes were identified using stability-based relative clustering validation on publicly available Mullen Scales of Early Learning (MSEL) and Vineland Adaptive Behavior Scales (VABS) data (n = 615; age = 24-68 months) from the National Institute of Mental Health Data Archive (NDA). Differential developmental trajectories between subtypes were tested on longitudinal data from NDA and from an independent in-house dataset from UCSD. A subset of the UCSD dataset was also tested for subtype differences in functional and structural neuroimaging phenotypes and relationships with blood gene expression. The current subtyping model was also compared to early language outcome subtypes derived from past work. RESULTS: Two autism subtypes can be identified based on early phenotypic LIMA features. These data-driven subtypes are robust in the population and can be identified in independent data with 98% accuracy. The subtypes can be described as Type I versus Type II autisms differentiated by relatively high versus low scores on LIMA features. These two types of autisms are also distinguished by different developmental trajectories over the first decade of life. Finally, these two types of autisms reveal striking differences in functional and structural neuroimaging phenotypes and their relationships with gene expression and may highlight unique biological mechanisms. LIMITATIONS: Sample sizes for the neuroimaging and gene expression dataset are relatively small and require further independent replication. The current work is also limited to subtyping based on MSEL and VABS phenotypic measures. CONCLUSIONS: This work emphasizes the potential importance of stratifying autism by a Type I versus Type II distinction focused on LIMA features and which may be of high prognostic and biological significance.

Epistasis is caused by genetic interactions among mutations that affect fitness. To characterize properties and potential mechanisms of epistasis, we engineered eight double mutants that combined mutations from the rho and rpoB genes of Escherichia coli. The two genes encode essential functions for transcription, and the mutations in each gene were chosen because they were beneficial for adaptation to thermal stress (42.2 °C). The double mutants exhibited patterns of fitness epistasis that included diminishing returns epistasis at 42.2 °C, stronger diminishing returns between mutations with larger beneficial effects and both negative and positive (sign) epistasis across environments (20.0 °C and 37.0 °C). By assessing gene expression between single and double mutants, we detected hundreds of genes with gene expression epistasis. Previous work postulated that highly connected hub genes in coexpression networks have low epistasis, but we found the opposite: hub genes had high epistasis values in both coexpression and protein-protein interaction networks. We hypothesized that elevated epistasis in hub genes reflected that they were enriched for targets of Rho termination but that was not the case. Altogether, gene expression and coexpression analyses revealed that thermal adaptation occurred in modules, through modulation of ribonucleotide biosynthetic processes and ribosome assembly, the attenuation of expression in genes related to heat shock and stress responses, and with an overall trend toward restoring gene expression toward the unstressed state.

Zinc (Zn) is a major soil contaminant and high Zn levels can disrupt growth, survival, and reproduction of fungi. Some fungal species evolved Zn tolerance through cell processes mitigating Zn toxicity, although the genes and detailed mechanisms underlying mycorrhizal fungal Zn tolerance remain unexplored. To fill this gap in knowledge, we investigated the gene expression of Zn tolerance in the ectomycorrhizal fungus Suillus luteus. We found that Zn tolerance in this species is mainly a constitutive trait that can also be environmentally dependent. Zinc tolerance in S. luteus is associated with differences in the expression of genes involved in metal exclusion and immobilization, as well as recognition and mitigation of metal-induced oxidative stress. Differentially expressed genes were predicted to be involved in transmembrane transport, metal chelation, oxidoreductase activity, and signal transduction. Some of these genes were previously reported as candidates for S. luteus Zn tolerance, while others are reported here for the first time. Our results contribute to understanding the mechanisms of fungal metal tolerance and pave the way for further research on the role of fungal metal tolerance in mycorrhizal associations.

In frontotemporal lobar degeneration (FTLD), pathological protein aggregation in specific brain regions is associated with declines in human-specialized social-emotional and language functions. In most patients, disease protein aggregates contain either TDP-43 (FTLD-TDP) or tau (FTLD-tau). Here, we explored whether FTLD-associated regional degeneration patterns relate to regional gene expression of human accelerated regions (HARs), conserved sequences that have undergone positive selection during recent human evolution. To this end, we used structural neuroimaging from patients with FTLD and human brain regional transcriptomic data from controls to identify genes expressed in FTLD-targeted brain regions. We then integrated primate comparative genomic data to test our hypothesis that FTLD targets brain regions linked to expression levels of recently evolved genes. In addition, we asked whether genes whose expression correlates with FTLD atrophy are enriched for genes that undergo cryptic splicing when TDP-43 function is impaired. We found that FTLD-TDP and FTLD-tau subtypes target brain regions with overlapping and distinct gene expression correlates, highlighting many genes linked to neuromodulatory functions. FTLD atrophy-correlated genes were strongly enriched for HARs. Atrophy-correlated genes in FTLD-TDP showed greater overlap with TDP-43 cryptic splicing genes and genes with more numerous TDP-43 binding sites compared with atrophy-correlated genes in FTLD-tau. Cryptic splicing genes were enriched for HAR genes, and vice versa, but this effect was due to the confounding influence of gene length. Analyses performed at the individual-patient level revealed that the expression of HAR genes and cryptically spliced genes within putative regions of disease onset differed across FTLD-TDP subtypes. Overall, our findings suggest that FTLD targets brain regions that have undergone recent evolutionary specialization and provide intriguing potential leads regarding the transcriptomic basis for selective vulnerability in distinct FTLD molecular-anatomical subtypes.

We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.

Genome analysis of individuals affected by retinitis pigmentosa (RP) identified two rare nucleotide substitutions at the same genomic location on chromosome 11 (g.61392563 [GRCh38]), 69 base pairs upstream of the start codon of the ciliopathy gene TMEM216 (c.-69G>A, c.-69G>T [GenBank: NM_001173991.3]), in individuals of South Asian and African ancestry, respectively. Genotypes included 71 homozygotes and 3 mixed heterozygotes in trans with a predicted loss-of-function allele. Haplotype analysis showed single-nucleotide variants (SNVs) common across families, suggesting ancestral alleles within the two distinct ethnic populations. Clinical phenotype analysis of 62 available individuals from 49 families indicated a similar clinical presentation with night blindness in the first decade and progressive peripheral field loss thereafter. No evident systemic ciliopathy features were noted. Functional characterization of these variants by luciferase reporter gene assay showed reduced promotor activity. Nanopore sequencing confirmed the lower transcription of the TMEM216 c.-69G>T allele in blood-derived RNA from a heterozygous carrier, and reduced expression was further recapitulated by qPCR, using both leukocytes-derived RNA of c.-69G>T homozygotes and total RNA from genome-edited hTERT-RPE1 cells carrying homozygous TMEM216 c.-69G>A. In conclusion, these variants explain a significant proportion of unsolved cases, specifically in individuals of African ancestry, suggesting that reduced TMEM216 expression might lead to abnormal ciliogenesis and photoreceptor degeneration.

Deciphering the genetic basis of prostate-specific antigen (PSA) levels may improve their utility for prostate cancer (PCa) screening. Using genome-wide association study (GWAS) summary statistics from 95,768 PCa-free men, we conducted a transcriptome-wide association study (TWAS) to examine impacts of genetically predicted gene expression on PSA. Analyses identified 41 statistically significant (p 0.5: CCNA2 and HIST1H2BN. Six of the 20 identified genes are not known to impact PCa risk. Fine-mapping based on whole blood and prostate tissue revealed five protein-coding genes with evidence of causal relationships with PSA levels. Of these five genes, four exhibited evidence of colocalization and one was conditionally independent of previous GWAS findings. These results yield hypotheses that should be further explored to improve understanding of genetic factors underlying PSA levels.

We created a 2-week, dual-module summer course introducing high school students to environmental toxicology by teaching them quantitative polymerase chain reaction (qPCR) as a way to quantify gene expression of chemical defense proteins in response to exposure to environmental pollutants. During the course, students are guided through the various stages of a successful qPCR experiment: in silico primer design and quality control, total RNA extraction and isolation, cDNA conversion, primer test PCR, and evaluation of results via agarose gel electrophoresis or UV/Vis spectra. The course combines lectures, discussions, and demonstrations with dry and wet laboratory sections to give students a thorough understanding of the scope, utility, and chemical principles of qPCR. At the end of the course, the students are taught how to analyze qPCR data and are encouraged to discuss their findings with other classmates to evaluate their hypotheses and assess possible sources of error. This course was designed to be easily adaptable to multiple test species, chemical exposures, and genes of interest. To explore both terrestrial and aquatic toxicology, the students use honey bees (Apis mellifera) and mosquitofish (Gambusia affinis) as test organisms, as well as ABC-type efflux transporters, antioxidant enzymes, and cytochrome P450 enzymes as endpoints for assessing gene expression. We share this course setup and applied protocols to encourage others to design and offer similar courses that give high school students a hands-on introduction to a broad swath of environmental toxicology research and an opportunity to develop scientific skills necessary for university-level research.

Aposematic organisms rely on their conspicuous appearance to signal that they are defended and unpalatable. Such phenotypes are strongly tied to survival and reproduction. Aposematic colors and patterns are highly variable; however, the genetic, biochemical, and physiological mechanisms producing this conspicuous coloration remain largely unidentified. Here, we identify genes potentially affecting color variation in two color morphs of Ranitomeya imitator: the orange-banded Sauce and the redheaded Varadero morphs. We examine gene expression in black and orange skin patches from the Sauce morph and black and red skin patches from the Varadero morph. We identified genes differentially expressed between skin patches, including those that are involved in melanin synthesis (e.g. mlana, pmel, tyrp1), iridophore development (e.g. paics, ppat, ak1), pteridine synthesis (e.g. gch1, pax3-a, xdh), and carotenoid metabolism (e.g. dgat2, rbp1, scarb2). In addition, using weighted correlation network analysis, we identified the top 50 genes with high connectivity from the most significant network associated with gene expression differences between color morphs. Of these 50 genes, 13 were known to be related to color production (gch1, gmps, gpr143, impdh1, mc1r, pax3-a, pax7, ppat, rab27a, rlbp1, tfec, trpm1, xdh).

BACKGROUND: Sex-differential biology may contribute to the consistently male-biased prevalence of autism spectrum disorder (ASD). Gene expression differences between males and females in the brain can indicate possible molecular and cellular mechanisms involved, although transcriptomic sex differences during human prenatal cortical development have been incompletely characterized, primarily due to small sample sizes. METHODS: We performed a meta-analysis of sex-differential expression and co-expression network analysis in 2 independent bulk RNA sequencing datasets generated from cortex of 273 prenatal donors without known neuropsychiatric disorders. To assess the intersection between neurotypical sex differences and neuropsychiatric disorder biology, we tested for enrichment of ASD-associated risk genes and expression changes, neuropsychiatric disorder risk genes, and cell type markers within identified sex-differentially expressed genes (sex-DEGs) and sex-differential co-expression modules. RESULTS: We identified 101 significant sex-DEGs, including Y-chromosome genes, genes impacted by X-chromosome inactivation, and autosomal genes. Known ASD risk genes, implicated by either common or rare variants, did not preferentially overlap with sex-DEGs. We identified 1 male-specific co-expression module enriched for immune signaling that is unique to 1 input dataset. CONCLUSIONS: Sex-differential gene expression is limited in prenatal human cortex tissue, although meta-analysis of large datasets allows for the identification of sex-DEGs, including autosomal genes that encode proteins involved in neural development. Lack of sex-DEG overlap with ASD risk genes in the prenatal cortex suggests that sex-differential modulation of ASD symptoms may occur in other brain regions, at other developmental stages, or in specific cell types, or may involve mechanisms that act downstream from mutation-carrying genes.

Extrachromosomal DNA is a common cause of oncogene amplification in cancer. The non-chromosomal inheritance of ecDNA enables tumors to rapidly evolve, contributing to treatment resistance and poor outcome for patients. The transcriptional context in which ecDNAs arise and progress, including chromosomally-driven transcription, is incompletely understood. We examined gene expression patterns of 870 tumors of varied histological types, to identify transcriptional correlates of ecDNA. Here, we show that ecDNA-containing tumors impact four major biological processes. Specifically, ecDNA-containing tumors up-regulate DNA damage and repair, cell cycle control, and mitotic processes, but down-regulate global immune regulation pathways. Taken together, these results suggest profound alterations in gene regulation in ecDNA-containing tumors, shedding light on molecular processes that give rise to their development and progression.

The cellular components of tumors and their microenvironment play pivotal roles in tumor progression, patient survival, and the response to cancer treatments. Unveiling a comprehensive cellular profile within bulk tumors via single-cell RNA sequencing (scRNA-seq) data is crucial, as it unveils intrinsic tumor cellular traits that elude identification through conventional cancer subtyping methods. Our contribution, scBeacon, is a tool that derives cell-type signatures by integrating and clustering multiple scRNA-seq datasets to extract signatures for deconvolving unrelated tumor datasets on bulk samples. Through the employment of scBeacon on the The Cancer Genome Atlas (TCGA) cohort, we find cellular and molecular attributes within specific tumor categories, many with patient outcome relevance. We developed a tumor cell-type map to visually depict the relationships among TCGA samples based on the cell-type inferences.

Quantitative susceptibility mapping (QSM) is an MRI modality used to non-invasively measure iron content in the brain. Iron exhibits a specific anatomically varying pattern of accumulation in the brain across individuals. The highest regions of accumulation are the deep grey nuclei, where iron is stored in paramagnetic molecule ferritin. This form of iron is considered to be what largely contributes to the signal measured by QSM in the deep grey nuclei. It is also known that QSM is affected by diamagnetic myelin contents. Here, we investigate spatial gene expression of iron and myelin related genes, as measured by the Allen Human Brain Atlas, in relation to QSM images of age-matched subjects. We performed multiple linear regressions between gene expression and the average QSM signal within 34 distinct deep grey nuclei regions. Our results show a positive correlation (p

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNAGly and BioRNALeu, respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.

Author(s): Ashok Kumar Saxena (corresponding author) [1]; Suman Choudhary [2]; Geetanjali Chilkote [1] Anaesthesiologists and pain physicians approach the genetics of pain management differently, intrigued by significant interindividual differences in [...]

Purpose: This study aimed to investigate the inhibitory effect of a PRG Barrier Coat on biofilm formation and structure by Streptococcus mutans and propose an effective method for preventing dental caries. Materials and Methods: Streptococcus mutans MT8148 biofilms were obtained from hydroxyapatite disks with and without a PRG Barrier Coat. Scanning electron microscopy (SEM) was used to observe the 12- and 24-h-cultured biofilms, while reverse-transcription polymerase chain reaction (qRT-PCR) was used to quantify caries-related genes. Biofilm adhesion assessments were performed on glass. Statistical analysis was performed using a two-sample t-test. Results: A statistically significant difference in Streptococcus mutans biofilm adhesion rate was observed between the control and PRG Barrier Coat-coated samples (p < 0.01). However, there was no statistically significant difference in total bacterial count or biofilm volume (p > 0.05). SEM revealed that the PRG Barrier Coat inhibited biofilm formation by Streptococcus mutans. Real-time RT-PCR revealed that the material restricted the expression of genes associated with caries-related biofilm formation. However, the suppression of gtfD and dexB differed from that of other genes. Conclusion: PRG Barrier Coat suppressed biofilm formation by Streptococcus mutans by inhibiting the expression of insoluble glucan synthase, which is associated with primary biofilm formation. The material also affected gene expression and altered the biofilm structure. Tooth surface-coating materials, such as PRG Barrier Coat, may improve caries prevention in dental practice. [ABSTRACT FROM AUTHOR]

Purpose: To screen for the cisplatin resistance-related prognostic signature in oral squamous cell carcinoma (OSCC) and assess its correlation with the immune microenvironment. Materials and Methods: The gene expression data associated with OSCC and cisplatin-resistance were downloaded from TCGA and GEO databases. Cisplatin-resistant genes were selected through taking the intersection of differentially expressed genes (DEGs) between tumor and control groups as well as between cisplatin-resistant samples and parental samples. Then, prognosis-related cisplatin-resistant genes were further selected by univariate Cox regression and LASSO regression analyses to construct a survival prognosis model. A GSEA (gene set enrichment analysis) between two risk groups was conducted with the MSigDB v7.1 database. Finally, the immune landscape of the sample was studied using CIBERSORT. The IC50 values of 57 drugs were predicted using pRRophetic 0.5. Results: A total 230 candidate genes were obtained. Then 7 drug-resistant genes were selected for prognostic risk-score (RS) signature construction using LASSO regression analysis, including STC2, TBC1D2, ADM, NDRG1, OLR1, PDGFA and ANO1. RS was an independent prognostic factor. Additionally, a nomogram model was established to predict the 1-, 2-, and 3-year survival rates of samples. The GSEA identified several differential pathways between two risk groups, such as EMT, hypoxia, and oxidative phosphorylation. Fifteen immune cells were statistically significantly different in infiltration level between the two groups, such as macrophages M2, and resting NK cells. A total of 57 drugs had statistically significantly different IC50 values between two risk groups. Conclusion: These model genes and immune cells may play a role in predicting the prognosis and chemoresistance in OSCC. [ABSTRACT FROM AUTHOR]

Macrophages regulate essential aspects of innate immunity against pathogens. In response to microbial components, macrophages activate primary and secondary inflammatory gene programs crucial for host defense. The liver X receptors (LXRα, LXRβ) are ligand-dependent nuclear receptors that direct gene expression important for cholesterol metabolism and inflammation, but little is known about the individual roles of LXRα and LXRβ in antimicrobial responses. Here, the results demonstrate that induction of LXRα transcription by prolonged exposure to lipopolysaccharide (LPS) supports inflammatory gene expression in macrophages. LXRα transcription is induced by NF-κB and type-I interferon downstream of TLR4 activation. Moreover, LPS triggers a reprogramming of the LXRα cistrome that promotes cytokine and chemokine gene expression through direct LXRα binding to DNA consensus sequences within cis-regulatory regions including enhancers. LXRα-deficient macrophages present fewer binding of p65 NF-κB and reduced histone H3K27 acetylation at enhancers of secondary inflammatory response genes. Mice lacking LXRα in the hematopoietic compartment show impaired responses to bacterial endotoxin in peritonitis models, exhibiting reduced neutrophil infiltration and decreased expansion and inflammatory activation of recruited F4/80lo-MHC-IIhi peritoneal macrophages. Together, these results uncover a previously unrecognized function for LXRα-dependent transcriptional cis-activation of secondary inflammatory gene expression in macrophages and the host response to microbial ligands.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Å resolution. The X-ray crystal structure complements recent cryo-EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage-cargo structures via X-ray crystallography. The results suggest the possibility that cryo-EM scaffolds may be repurposed for structure determination by X-ray crystallography, thus extending the utility of electron-microscopy scaffold designs for alternative structural biology applications.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the WIN site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

The conformational state of DNA fine-tunes the transcriptional rate and abundance of RNA. Here, we report that G-quadruplex DNA (G4-DNA) accumulates in neurons, in an experience-dependent manner, and that this is required for the transient silencing and activation of genes that are critically involved in learning and memory in male C57/BL6 mice. In addition, site-specific resolution of G4-DNA by dCas9-mediated deposition of the helicase DHX36 impairs fear extinction memory. Dynamic DNA structure states therefore represent a key molecular mechanism underlying memory consolidation.One-Sentence Summary: G4-DNA is a molecular switch that enables the temporal regulation of the gene expression underlying the formation of fear extinction memory.

PurposeOne plausible mechanistic hypothesis is the potential contribution of inflammatory mechanisms to shortness of breath. This study was aimed to evaluate for associations between the occurrence of shortness of breath and perturbations in inflammatory pathways.MethodsPatients with cancer reported the occurrence of shortness of breath six times over two cycles of chemotherapy. Latent class analysis was used to identify subgroups of patients with distinct shortness of breath occurrence profiles (i.e., none (70.5%), decreasing (8.2%), increasing (7.8%), high (13.5%)). Using an extreme phenotype approach, whole transcriptome differential gene expression and pathway impact analyses were performed to evaluate for perturbed signaling pathways associated with shortness of breath between the none and high classes. Two independent samples (RNA-sequencing (n = 293) and microarray (n = 295) methodologies) were evaluated. Fisher's combined probability method was used to combine these results to obtain a global test of the null hypothesis. In addition, an unweighted knowledge network was created using the specific pathway maps to evaluate for interconnections among these pathways.ResultsTwenty-nine Kyoto Encyclopedia of Genes and Genomes inflammatory signaling pathways were perturbed. The mitogen-activated protein kinase signaling pathway node had the highest closeness, betweenness, and degree scores. In addition, five common respiratory disease-related pathways, that may share mechanisms with cancer-related shortness of breath, were perturbed.ConclusionsFindings provide preliminary support for the hypothesis that inflammation contribute to the occurrence of shortness of breath in patients with cancer. In addition, the mechanisms that underlie shortness of breath in oncology patients may be similar to other respiratory diseases.

In animal models, Nipbl deficiency phenocopies gene expression changes and birth defects seen in Cornelia de Lange syndrome, the most common cause of which is Nipbl haploinsufficiency. Previous studies in Nipbl+/- mice suggested that heart development is abnormal as soon as cardiogenic tissue is formed. To investigate this, we performed single-cell RNA sequencing on wild-type and Nipbl+/- mouse embryos at gastrulation and early cardiac crescent stages. Nipbl+/- embryos had fewer mesoderm cells than wild-type and altered proportions of mesodermal cell subpopulations. These findings were associated with underexpression of genes implicated in driving specific mesodermal lineages. In addition, Nanog was found to be overexpressed in all germ layers, and many gene expression changes observed in Nipbl+/- embryos could be attributed to Nanog overexpression. These findings establish a link between Nipbl deficiency, Nanog overexpression, and gene expression dysregulation/lineage misallocation, which ultimately manifest as birth defects in Nipbl+/- animals and Cornelia de Lange syndrome.

Regulation of transcription and translation are mechanisms through which genetic variants affect complex traits. Expression quantitative trait locus (eQTL) studies have been more successful at identifying cis-eQTL (within 1 Mb of the transcription start site) than trans-eQTL. Here, we tested the cis component of gene expression for association with observed plasma protein levels to identify cis- and trans-acting genes that regulate protein levels. We used transcriptome prediction models from 49 Genotype-Tissue Expression (GTEx) Project tissues to predict the cis component of gene expression and tested the predicted expression of every gene in every tissue for association with the observed abundance of 3,622 plasma proteins measured in 3,301 individuals from the INTERVAL study. We tested significant results for replication in 971 individuals from the Trans-omics for Precision Medicine (TOPMed) Multi-Ethnic Study of Atherosclerosis (MESA). We found 1,168 and 1,210 cis- and trans-acting associations that replicated in TOPMed (FDR

A new in vitro system called Rec-Seq sheds light on how mRNA molecules compete for the machinery that translates their genetic sequence into proteins.

In some patients with myeloproliferative neoplasms (MPN), two genetic mutations are often found: JAK2 V617F and one in the TET2 gene. Whether one mutation is present influences how the other subsequent mutation will affect the regulation of gene expression. In other words, when a patient carries both mutations, the order of when they first arose has been shown to influence disease progression and prognosis. We propose a nonlinear ordinary differential equation, the Moran process, and Markov chain models to explain the non-additive and non-commutative mutation effects on recent clinical observations of gene expression patterns, proportions of cells with different mutations, and ages at diagnosis of MPN. Combined, these observations are used to shape our modeling framework. Our key proposal is that bistability in gene expression provides a natural explanation for many observed order-of-mutation effects. We also propose potential experimental measurements that can be used to confirm or refute predictions of our models.

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozymes self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the Cpeb3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.

Genome-wide association studies (GWASs) have uncovered susceptibility loci associated with psychiatric disorders such as bipolar disorder (BP) and schizophrenia (SCZ). However, most of these loci are in non-coding regions of the genome, and the causal mechanisms of the link between genetic variation and disease risk is unknown. Expression quantitative trait locus (eQTL) analysis of bulk tissue is a common approach used for deciphering underlying mechanisms, although this can obscure cell-type-specific signals and thus mask trait-relevant mechanisms. Although single-cell sequencing can be prohibitively expensive in large cohorts, computationally inferred cell-type proportions and cell-type gene expression estimates have the potential to overcome these problems and advance mechanistic studies. Using bulk RNA-seq from 1,730 samples derived from whole blood in a cohort ascertained from individuals with BP and SCZ, this study estimated cell-type proportions and their relation with disease status and medication. For each cell type, we found between 2,875 and 4,629 eGenes (genes with an associated eQTL), including 1,211 that are not found on the basis of bulk expression alone. We performed a colocalization test between cell-type eQTLs and various traits and identified hundreds of associations that occur between cell-type eQTLs and GWASs but that are not detected in bulk eQTLs. Finally, we investigated the effects of lithium use on the regulation of cell-type expression loci and found examples of genes that are differentially regulated according to lithium use. Our study suggests that applying computational methods to large bulk RNA-seq datasets of non-brain tissue can identify disease-relevant, cell-type-specific biology of psychiatric disorders and psychiatric medication.

Generalist microbes have adapted to a multitude of environmental stresses through their integrated stress response system. Individual stress responses have been quantified by E. coli metabolism and expression (ME) models under thermal, oxidative and acid stress, respectively. However, the systematic quantification of cross-stress & cross-talk among these stress responses remains lacking. Here, we present StressME: the unified stress response model of E. coli combining thermal (FoldME), oxidative (OxidizeME) and acid (AcidifyME) stress responses. StressME is the most up to date ME model for E. coli and it reproduces all published single-stress ME models. Additionally, it includes refined rate constants to improve prediction accuracy for wild-type and stress-evolved strains. StressME revealed certain optimal proteome allocation strategies associated with cross-stress and cross-talk responses. These stress-optimal proteomes were shaped by trade-offs between protective vs. metabolic enzymes; cytoplasmic vs. periplasmic chaperones; and expression of stress-specific proteins. As StressME is tuned to compute metabolic and gene expression responses under mild acid, oxidative, and thermal stresses, it is useful for engineering and health applications. The modular design of our open-source package also facilitates model expansion (e.g., to new stress mechanisms) by the computational biology community.

Small-molecule control of gene expression underlies the function of numerous engineered gene circuits that are capable of environmental sensing, computation, and memory. While many recently developed inducible promoters have been tailor-made for bacteria or mammalian cells, relatively few new systems have been built for Saccharomyces cerevisiae, limiting the scale of synthetic biology work that can be done in yeast. To address this, we created the yeast Tunable Expression Systems Toolkit (yTEST), which contains a set of five extensively characterized inducible promoter systems regulated by the small-molecules doxycycline (Dox), abscisic acid (ABA), danoprevir (DNV), 1-naphthaleneacetic acid (NAA), and 5-phenyl-indole-3-acetic acid (5-Ph-IAA). Assembly was made to be compatible with the modular cloning yeast toolkit (MoClo-YTK) to enhance the ease of use and provide a framework to benchmark and standardize each system. Using this approach, we built multiple systems with maximal expression levels greater than those of the strong constitutive TDH3 promoter. Furthermore, each of the five classes of systems could be induced at least 60-fold after a 6 h induction and the highest fold change observed was approximately 300. Thus, yTEST provides a reliable, diverse, and customizable set of inducible promoters to modulate gene expression in yeast for applications in synthetic biology, metabolic engineering, and basic research.

BACKGROUND: How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum have long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdés, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. RESULTS: The final assembled and filtered Berghia genome is comparable to other high-quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes) and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. CONCLUSIONS: Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

BackgroundStatins lower circulating low-density lipoprotein cholesterol (LDLC) levels and reduce cardiovascular disease risk. Though highly efficacious in general, there is considerable inter-individual variation in statin efficacy that remains largely unexplained.MethodsTo identify novel genes that may modulate statin-induced LDLC lowering, we used RNA-sequencing data from 426 control- and 2 µM simvastatin-treated lymphoblastoid cell lines (LCLs) derived from European and African American ancestry participants of the Cholesterol and Pharmacogenetics (CAP) 40 mg/day 6-week simvastatin clinical trial (ClinicalTrials.gov Identifier: NCT00451828). We correlated statin-induced changes in LCL gene expression with plasma LDLC statin response in the corresponding CAP participants. For the most correlated gene identified (ZNF335), we followed up in vivo by comparing plasma cholesterol levels, lipoprotein profiles, and lipid statin response between wild-type mice and carriers of a hypomorphic (partial loss of function) missense mutation in Zfp335 (the mouse homolog of ZNF335).ResultsThe statin-induced expression changes of 147 human LCL genes were significantly correlated to the plasma LDLC statin responses of the corresponding CAP participants in vivo (FDR = 5%). The two genes with the strongest correlations were zinc finger protein 335 (ZNF335 aka NIF-1, rho = 0.237, FDR-adj p = 0.0085) and CCR4-NOT transcription complex subunit 3 (CNOT3, rho = 0.233, FDR-adj p = 0.0085). Chow-fed mice carrying a hypomorphic missense (R1092W; aka bloto) mutation in Zfp335 had significantly lower non-HDL cholesterol levels than wild-type C57BL/6J mice in a sex combined model (p = 0.04). Furthermore, male (but not female) mice carrying the Zfp335R1092W allele had significantly lower total and HDL cholesterol levels than wild-type mice. In a separate experiment, wild-type mice fed a control diet for 4 weeks and a matched simvastatin diet for an additional 4 weeks had significant statin-induced reductions in non-HDLC (-43 ± 18% and -23 ± 19% for males and females, respectively). Wild-type male (but not female) mice experienced significant reductions in plasma LDL particle concentrations, while male mice carrying Zfp335R1092W allele(s) exhibited a significantly blunted LDL statin response.ConclusionsOur in vitro and in vivo studies identified ZNF335 as a novel modulator of plasma cholesterol levels and statin response, suggesting that variation in ZNF335 activity could contribute to inter-individual differences in statin clinical efficacy.

Friedreichs ataxia is a neurodegenerative disorder caused by reduced frataxin levels. It leads to motor and sensory impairments and has a median life expectancy of around 35 years. As the most common inherited form of ataxia, Friedreichs ataxia lacks reliable, non-invasive biomarkers, prolonging and inflating the cost of clinical trials. This study proposes TUG1, a long non-coding RNA, as a promising blood-based biomarker for Friedreichs ataxia, which is known to regulate various cellular processes. In a previous study using a frataxin knockdown mouse model, we observed several hallmark Friedreichs ataxia symptoms. Building on this, we hypothesized that a dual-source approach-comparing the data from peripheral blood samples from Friedreichs ataxia patients with tissue samples from affected areas in Friedreichs ataxia knockdown mice, tissues usually unattainable from patients-would effectively identify robust biomarkers. A comprehensive reanalysis was conducted on gene expression data from 183 age- and sex-matched peripheral blood samples of Friedreichs ataxia patients, carriers and controls and 192 tissue data sets from Friedreichs ataxia knockdown mice. Blood and tissue samples underwent RNA isolation and quantitative reverse transcription polymerase chain reaction, and frataxin knockdown was confirmed through enzyme-linked immunosorbent assays. Tug1 RNA interaction was explored via RNA pull-down assays. Validation was performed in serum samples on an independent set of 45 controls and 45 Friedreichs ataxia patients and in blood samples from 66 heterozygous carriers and 72 Friedreichs ataxia patients. Tug1 and Slc40a1 emerged as potential blood-based biomarkers, confirmed in the Friedreichs ataxia knockdown mouse model (one-way ANOVA, P ≤ 0.05). Tug1 was consistently downregulated after Fxn knockdown and correlated strongly with Fxn levels (R 2 = 0.71 during depletion, R 2 = 0.74 during rescue). Slc40a1 showed a similar but tissue-specific pattern. Further validation of Tug1s downstream targets strengthened its biomarker candidacy. In additional human samples, TUG1 levels were significantly downregulated in both whole blood and serum of Friedreichs ataxia patients compared with controls (Wilcoxon signed-rank test, P < 0.05). Regression analyses revealed a negative correlation between TUG1 fold-change and disease onset (P < 0.0037) and positive correlations with disease duration and functional disability stage score (P < 0.04). This suggests that elevated TUG1 levels correlate with earlier onset and more severe cases. This study identifies TUG1 as a potential blood-based biomarker for Friedreichs ataxia, showing consistent expression variance in human and mouse tissues related to disease severity and key Friedreichs ataxia pathways. It correlates with frataxin levels, indicating its promise as an early, non-invasive marker. TUG1 holds potential for Friedreichs ataxia monitoring and therapeutic development, meriting additional research.

BackgroundStructural variations (SVs) have significant impacts on complex phenotypes by rearranging large amounts of DNA sequence.ResultsWe present a comprehensive SV catalog based on the whole-genome sequence of 1060 pigs (Sus scrofa) representing 101 breeds, covering 9.6% of the pig genome. This catalog includes 42,487 deletions, 37,913 mobile element insertions, 3308 duplications, 1664 inversions, and 45,184 break ends. Estimates of breed ancestry and hybridization using genotyped SVs align well with those from single nucleotide polymorphisms. Geographically stratified deletions are observed, along with known duplications of the KIT gene, responsible for white coat color in European pigs. Additionally, we identify a recent SINE element insertion in MYO5A transcripts of European pigs, potentially influencing alternative splicing patterns and coat color alterations. Furthermore, a Yorkshire-specific copy number gain within ABCG2 is found, impacting chromatin interactions and gene expression across multiple tissues over a stretch of genomic region of ~200 kb. Preliminary investigations into SV's impact on gene expression and traits using the Pig Genotype-Tissue Expression (PigGTEx) data reveal SV associations with regulatory variants and gene-trait pairs. For instance, a 51-bp deletion is linked to the lead eQTL of the lipid metabolism regulating gene FADS3, whose expression in embryo may affect loin muscle area, as revealed by our transcriptome-wide association studies.ConclusionsThis SV catalog serves as a valuable resource for studying diversity, evolutionary history, and functional shaping of the pig genome by processes like domestication, trait-based breeding, and adaptive evolution.

Plant growth requires the integration of internal and external cues, perceived and transduced into a developmental programme of cell division, elongation and wall thickening. Mechanical forces contribute to this regulation, and thigmomorphogenesis typically includes reducing stem height, increasing stem diameter, and a canonical transcriptomic response. We present data on a bZIP transcription factor involved in this process in grasses. Brachypodium distachyon SECONDARY WALL INTERACTING bZIP (SWIZ) protein translocated into the nucleus following mechanostimulation. Classical touch-responsive genes were upregulated in B. distachyon roots following touch, including significant induction of the glycoside hydrolase 17 family, which may be unique to grass thigmomorphogenesis. SWIZ protein binding to an E-box variant in exons and introns was associated with immediate activation followed by repression of gene expression. SWIZ overexpression resulted in plants with reduced stem and root elongation. These data further define plant touch-responsive transcriptomics and physiology, offering insights into grass mechanotranduction dynamics.

Spatially resolved transcriptomics technologies have opened new avenues for understanding gene expression heterogeneity in spatial contexts. However, existing methods for identifying spatially variable genes often focus solely on statistical significance, limiting their ability to capture continuous expression patterns and integrate spot-level covariates. To address these challenges, we introduce spVC, a statistical method based on a generalized Poisson model. spVC seamlessly integrates constant and spatially varying effects of covariates, facilitating comprehensive exploration of gene expression variability and enhancing interpretability. Simulation and real data applications confirm spVC's accuracy in these tasks, highlighting its versatility in spatial transcriptomics analysis.

The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

BackgroundGene expression may be regulated by the DNA methylation of regulatory elements in cis, distal, and trans regions. One method to evaluate the relationship between DNA methylation and gene expression is the mapping of expression quantitative trait methylation (eQTM) loci (also called expression associated CpG loci, eCpG). However, no open-source tools are available to provide eQTM mapping. In addition, eQTM mapping can involve a large number of comparisons which may prevent the analyses due to limitations of computational resources. Here, we describe Torch-eCpG, an open-source tool to perform eQTM mapping that includes an optimized implementation that can use the graphical processing unit (GPU) to reduce runtime.ResultsWe demonstrate the analyses using the tool are reproducible, up to 18 × faster using the GPU, and scale linearly with increasing methylation loci.ConclusionsTorch-eCpG is a fast, reliable, and scalable tool to perform eQTM mapping. Source code for Torch-eCpG is available at https://github.com/kordk/torch-ecpg .

Aging and cellular senescence are increasingly recognized as key contributors to pulmonary fibrosis. However, our understanding in the context of scleroderma-associated interstitial lung disease (SSc-ILD) is limited. To investigate, we leveraged previously established lung aging- and cell-specific senescence signatures to determine their presence and potential relevance to SSc-ILD. We performed a gene expression meta-analysis of lung tissues from 38 SSc-ILD and 18 healthy controls and found that markers (GDF15, COMP, and CDKN2A) and pathways (p53) of senescence were significantly increased in SSc-ILD. When probing the established aging and cellular senescence signatures, we found that epithelial and fibroblast senescence signatures had a 3.6- and 3.7-fold enrichment, respectively, in the lung tissue of SSc-ILD and that lung aging genes (CDKN2A, FRZB, PDE1A, and NAPI12) were increased in SSc-ILD. These signatures were also enriched in SSc skin and associated with degree of skin involvement (limited vs. diffuse cutaneous). To further support these findings, we examined telomere length (TL), a surrogate for aging, in the lung tissue and found that, independent of age, SSc-ILD had significantly shorter telomeres than controls in type II alveolar cells in the lung. TL in SSc-ILD was comparable to idiopathic pulmonary fibrosis, a disease of known aberrant aging. Taken together, this study provides novel insight into the possible mechanistic effects of accelerated aging and aberrant cellular senescence in SSc-ILD pathogenesis.

Aging-related memory impairment and pathological memory disorders such as Alzheimers disease differ between males and females, and yet little is known about how aging-related changes in the transcriptome and chromatin environment differ between sexes in the hippocampus. To investigate this question, we compared the chromatin accessibility landscape and gene expression/alternative splicing pattern of young adult and aged mouse hippocampus in both males and females using ATAC-seq and RNA-seq. We detected significant aging-dependent changes in the expression of genes involved in immune response and synaptic function and aging-dependent changes in the alternative splicing of myelin sheath genes. We found significant sex-bias in the expression and alternative splicing of hundreds of genes, including aging-dependent female-biased expression of myelin sheath genes and aging-dependent male-biased expression of genes involved in synaptic function. Aging was associated with increased chromatin accessibility in both male and female hippocampus, especially in repetitive elements, and with an increase in LINE-1 transcription. We detected significant sex-bias in chromatin accessibility in both autosomes and the X chromosome, with male-biased accessibility enriched at promoters and CpG-rich regions. Sex differences in gene expression and chromatin accessibility were amplified with aging, findings that may shed light on sex differences in aging-related and pathological memory loss.

BackgroundCost-effective production of biofuels from lignocellulose requires the fermentation of D-xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD+ to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments.ResultsWe screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae. Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis.ConclusionCollectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

Chlamydomonas reinhardtii , a unicellular green alga, is a prominent model for green biotechnology and for studying organelles' function and biogenesis, such as chloroplasts and cilia. However, the stable expression of foreign genes from the nuclear genome in C. reinhardtii faces several limitations, including low expression levels and significant differences between clones due to genome position effects, epigenetic silencing, and time-consuming procedures. We developed a robust transient expression system in C. reinhardtii to overcome these limitations. We demonstrated efficient entry of in vitro-transcribed mRNA into wall-less cells and enzymatically dewalled wild-type cells via electroporation. The endogenous or exogenous elements can facilitate efficient transient expression of mRNA in C. reinhardtii , including the 5' UTR of PsaD and the well-characterized Kozak sequence derived from the Chromochloris zofingiensis. In the optimized system, mRNA expression was detectable in 120 h with a peak around 4 h after transformation. Fluorescently tagged proteins were successfully transiently expressed, enabling organelle labeling and real-time determination of protein sub-cellular localization. Remarkably, transiently expressed IFT46 compensated for the ift46–1 mutant phenotype, indicating the correct protein folding and function of IFT46 within the cells. Additionally, we demonstrated the feasibility of our system for studying protein-protein interactions in living cells using bimolecular fluorescence complementation. In summary, the established transient expression system provides a powerful tool for investigating protein localization, function, and interactions in C. reinhardtii within a relatively short timeframe, which will significantly facilitate the study of gene function, genome structure, and green biomanufacturing in C. reinhardtii and potentially in other algae. • Successful establishment of an RNA-based transient expression system in Chlamydomonas. • Function of 5' UTR on gene expression occurs at the post-transcriptional level in Chlamydomonas. • Analysis of the localization, function, and interactions of proteins can be completed within one week. [ABSTRACT FROM AUTHOR]

Engineering of extracellular vesicles (EVs) towards more efficient targeting and uptake to specific cells has large potentials for their application as therapeutics. Carbohydrates play key roles in various biological interactions and are essential for EV biology. The extent to which glycan modification of EVs can be achieved through genetic glycoengineering of their parental cells has not been explored yet. Here we introduce targeted glycan modification of EVs through cell-based glycoengineering via modification of various enzymes in the glycosylation machinery. In a "simple cell" strategy, we modified major glycosylation pathways by knocking-out (KO) essential genes for N-glycosylation (MGAT1), O-GalNAc glycosylation (C1GALT1C1), glycosphingolipids (B4GALT5/6), glycosaminoglycans (B4GALT7) and sialylation (GNE) involved in the elongation or biosynthesis of the glycans in HEK293F cells. The gene editing led to corresponding glycan changes on the cells as demonstrated by differential lectin staining. Small EVs (sEVs) isolated from the cells showed overall corresponding glycan changes, but also some unexpected differences to their parental cell including enrichment preference for certain glycan structures and absence of other glycan types. The genetic glycoengineering did not significantly impact sEVs production, size distribution, or syntenin-1 biomarker expression, while a clonal influence on sEVs production yields was observed. Our findings demonstrate the successful implementation of sEVs glycoengineering via genetic modification of the parental cell and a stable source for generation of glycoengineered sEVs. The utilization of glycoengineered sEVs offers a promising opportunity to study the role of glycosylation in EV biology, as well as to facilitate the optimization of sEVs for therapeutic purposes. • Glycan modification of sEVs can be accomplished by genetic glycoengineering the cells from which they originate. • Glycan alterations on sEVs are not always in line with those found on their parental cells. • Glycoengineering of cells does not significantly affect the production, size, or expression of biomarkers on sEVs. • Cell-based glycoengineering enables the stable large-scale production of glycoengineered sEVs. [ABSTRACT FROM AUTHOR]

The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant "dead" MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in K. phaffii to regulate the expression of the enhanced green fluorescent protein (eGFP). A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of eGFP in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in K. phaffii through successfully regulating eGFP expression. • We report the first use of dMAD7 in K. phaffii to regulate the expression of enhanced green fluorescent protein (eGFP). • A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. • Whole genome sequencing and RNA-sequencing were used to assess the integration events and to characterize the strains. • Our findings conclusively demonstrate the utility of the dMAD7 system in K. phaffii. [ABSTRACT FROM AUTHOR]

This study highlights the significance of overexpressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from the MEP (methylerythritol 4-phosphate) pathway, in addition to short-chain prenyltransferase fusions for the improved production of the diterpene, taxa-4,11-diene, the first committed intermediate in the production of anti-cancer drug paclitaxel. The results showed that the strain which has (i) the taxadiene synthase (txs) gene integrated into the genome, (ii) the MEP pathway genes overexpressed, (iii) the fpps-crtE prenyltransferases fusion protein and (iv) additional expression of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), yielded the highest production of taxa-4,11-diene at 390 mg/L (26 mg/L/OD 600). This represents a thirteen-fold increase compared to the highest reported concentration in B. subtilis. The focus on additional overexpression of DXS and utilizing short-chain prenyltransferase fusions underscores their pivotal role in achieving significant titer improvements in terpene biosynthesis. [Display omitted] • Utilizing Bacillus subtilis as a cell factory for the production of taxadiene. • Additional expression of DXS from MEP pathway can boost taxadiene production. • Application of fused prenyltransferases can enhance the yield of taxadiene. • The highest taxadiene production so far in B. subtilis has been achieved. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells represent the most preferential host cell system for therapeutic monoclonal antibody (mAb) production. Enhancing mAb production in CHO cells can be achieved by adding chemical compounds that regulate the cell cycle and cell survival pathways. This study investigated the impact of ectoine supplementation on mAb production in CHO cells. The results showed that adding ectoine at a concentration of 100 mM on the 3rd day of cultivation improved mAb production by improving cell viability and extending the culture duration. RNA sequencing analysis revealed differentially expressed genes associated with cell cycle regulation, cell proliferation, and cellular homeostasis, in particular promotion of cell cycle arrest, which was then confirmed by flow cytometry analysis. Ectoine-treated CHO cells exhibited an increase in the number of cells in the G0/G1 phase. In addition, the cell diameter was also increased. These findings support the hypothesis that ectoine enhances mAb production in CHO cells through mechanisms involving cell cycle arrest and cellular homeostasis. Overall, this study highlights the potential of ectoine as a promising supplementation strategy to enhance mAb production not only in CHO cells but also in other cell lines. [Display omitted] • Ectoine improved mAb production in CHO cells. • Ectoine improved cell viability and extended the culture duration of CHO cells. • Ectoine increased the number of CHO cells in the G0/G1 phase. • Enhanced mAb production involved cell cycle arrest and cellular homeostasis. • Ectoine is a promising supplement to enhance mAb production in cell lines. [ABSTRACT FROM AUTHOR]